Excessive activation of ionotropic glutamate receptors increases oxidative stress, which contributes to the neurodegeneration observed following neurological insults such as ischemia and seizures, as well as contributes to neuronal death in neurodegenerative diseases (Alzheimer's, Parkinson's, etc.). From a clinical perspective, it is a clear threat to brain function and to survival. It is believed that generation of reactive oxygen species and ensuing oxidative stress is a major contributor to glutamate toxicity. At the same time, oxidative stress is a major cause of DNA damage, which is also a common component of neuronal injury. DNA damage may contribute to neuronal loss and injury not only after acute brain insults but also under various chronic neurodegenerative conditions, such as Alzheimer's, Huntington's, and Parkinson's diseases, amyotrophic lateral sclerosis, ataxia telangiectasia and many other neurological disorders. The most lethal form of DNA damage, the double strand breaks (DSBs), and the ability of cells to repair them has not yet been directly demonstrated following excessive stimulation of glutamate receptors. While limited evidence suggests the importance of DSBs and their repair machinery in vulnerability to glutamate-induced injury, no systematic direct studies have been done in mature neurons. We have developed a sensitive model to start addressing the role of DSB DNA damage in neuronal vulnerability to glutamate-mediated insults using phosphorylation of histone variant H2A.X, which occurs rapidly following DNA DSBs. Our general working hypothesis is that the consequences of unrepaired DSBs in terminally differentiated neurons are critical contributors to neuronal demise in the aftermath of excessive excitation. Conversely, successful repair of these breaks may increase neuronal survival following glutamate-driven insults. Specific Aims will test the following specific hypotheses aiming at proving this concept: 1) Increased phosphorylation of histone H2AX following activation of ionotropic glutamate receptors will result in increased DSB repair;this hypothesis we will tested by measuring DSB repair activity in rat cortical neuronal cultures;2) Impairment of H2AX phosphorylation will result in increased glutamate toxicity due to the disruption of the DSB repair pathway. To test this, we will examine vulnerability of neurons from H2AX-/- transgenic mice to vulnerability to glutamate toxicity and evaluate their DSB repair capabilities. We expect that H2AX-/- neurons will be more vulnerable to glutamate toxicity and demonstrate diminished DSB repair as compared to wild-type cells. Moreover, we will reconstitute functional histone H2AX in H2AX-/- neurons using lentiviral expression and evaluate the restoration of their resistance to glutamate toxicity. Testing these hypotheses may reveal a novel common mechanism contributing to neurotoxicity in a variety of neurodegenerative disorders, will lead to identification of attractive new targets for therapy of these disorders, and will lay a foundation for future interventional studies in vivo targeting DSB repair pathway in neurons. PUBLIC HEALTH RELEVANCE: Damage to DNA is a common component of neuronal injury. It may contribute to neuronal loss and injury not only after acute brain insult (e.g., prolonged seizures, stroke, TBI) but also under various chronic neurodegenerative conditions, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia telangiectasia, among other neurological disorders that currently have no effective cure. Excessive excitation also contributes to many of these pathologies and is believed to be the major cause of DNA damage. However, little is known about the mechanisms responsible for the excitation- driven formation of the most lethal type of DNA damage (double strand breaks) in neurons and the ability of nerve cells to withstand this damage. This proposal will examine these mechanisms and will lay the foundation for identification of new targets for therapy of a broad variety of neurological conditions relevant to excitotoxicity.